SRSV (Small Round Structured Virus) is commonly known as a causative virus of viral food poisoning. The present invention relates to nucleic acid sequences, oligonucleotides and method for detection of SRSV and, in particular, a virus which belongs to Genotype II (GII) in clinical examinations, public health examinations, food evaluations and food poisoning examinations.
SRSV belongs to the human Calicivirus group. Human Caliciviruses are classified according to their three genetic types: Genogroup I (GI), Genogroup II (GII) and Genogroup III (GIII). Generally speaking, GI and GII Caliciviruses are generally referred to as SRSV, and GIII Caliciviruses are referred to as human Caliciviruses in the narrow sense.
Approximately 20% of the food poisoning cases reported in Japan are attributed to viral causes. SRSV is detected in over 80% of these viral food poisoning cases. The major source of infection is food, and raw oysters are often implicated. SRSV has also been detected in infant (sporadic) acute enterogastritis, thus suggesting the possibility of propagation from human to human. SRSV detection therefore provides an important contribution to public health and food quality.
To date, SRSV detection has been relied on electron microscope observation. Detection by this method, however, requires the virus to be present in an amount of 106/ml or greater, and thus the detection subject was limited to patient""s feces. Further, even though observation of the virus was possible, it could not be identified.
In recent years, it has become possible to produce viroid hollow particles for human caliciviruses, and research is advancing toward a specific antibody-detecting ELISA employing such particles. However, the detection sensitivity is still on the same level as electron microscopy, and the method is therefore far from highly sensitive.
As mentioned above, since a complex procedure and a long time are required for the conventional method and it is difficult to detect trace amounts of SRSV in samples within a short time, it has been desired to provide a detection method satisfying the high-speed and high-sensitivity requirements for food evaluation and the like. There has also been a demand for development of an automated examination device which allows more convenient examination.
Methods of amplifying target nucleic acid can be utilized as highly sensitive detection methods. One known method for amplification of specific sequences of genomic RNA such as that of SRSV is the reverse transcription-polymerase chain reaction (RT-PCR). This method comprises synthesis of a cDNA for the target RNA by a reverse transcription step, and then repeating a cycle of heat denaturation, primer annealing and extension reaction in the presence of a pair of primers which are complementary and homologous to both ends of specific sequences of the cDNA (the antisense primer may be the one used in the reverse transcription step) as well as a thermostable DNA polymerase, thereby amplifying the specific DNA sequence. However, the RT-PCR method requires a two-step procedure (a reverse transcription step and a PCR step), as well as a procedure involving rapidly increasing and decreasing the temperature, which prevent its automation.
Other methods known for amplification of specific RNA sequences include the NASBA and 3SR methods which accomplish amplification of specific RNA sequences by the concerted action of reverse transcriptase and RNA polymerase. In these methods, the target RNA is used as a template in the synthesis of a promoter sequence-containing double-stranded DNA using a promoter sequence-containing primer, reverse transcriptase and Ribonuclease H; this double-stranded DNA provides a template in the synthesis of an RNA containing the specific base sequence of the target RNA using an RNA polymerase; subsequently, this RNA provides a template in a chain reaction for synthesizing a double-stranded DNA containing the promoter sequence.
Thus, the NASBA and 3SR methods allow nucleic acid amplification at a constant temperature and are therefore considered suitable for automation. However, as these amplification methods involve relatively low temperature reactions (41xc2x0 C., for example), the target RNA forms an intramolecular structure which inhibits binding of the primer and may reduce the reaction efficiency. Therefore, they require subjecting the target RNA to heat denaturation before the amplification reaction so as to destroy the intramolecular structure of the target RNA and thus to improve the primer binding efficiency. Further, even when carrying out the detection of an RNA at a lower temperature, these methods require an oligonucleotide capable of binding to the RNA forming such a molecular structure.
Thus, an object of the present invention is to provide nucleic acid sequences, oligonucleotides or suitable combination thereof, capable of specifically cleaving or amplifying SRSV and, in particular, a virus which belongs to GII type, preferably at a relatively low and constant temperature (between 35xc2x0 C. and 50xc2x0 C., preferably 41xc2x0 C.), useful in detecting and identifying such a virus at high sensitivity.
The invention of claim 1, which has been accomplished to achieve this object, relates to a cDNA as shown in SEQ. ID. No.1, or fragment or derivative thereof having a size sufficient to bind to Genogroup II type Small Round Structured Virus (SRSV).
The invention of claim 2, which has been accomplished to achieve the aforementioned object, relates to an oligonucleotide for detection of GII type SRSV, which oligonucleotide is capable of binding to said GII type SRSV at specific site, and comprises at least 10 contiguous bases of any of the sequences listed as SEQ. ID. Nos.2 to 9.
The invention of claim 3, which has been accomplished to achieve the aforementioned object, relates to the oligonucleotide according to claim 2, wherein said oligonucleotide is an oligonucleotide probe for cleaving said RNA at said specific site by binding to said specific site of said RNA.
The invention of claim 4, which has been accomplished to achieve the aforementioned object, relates to the oligonucleotide according to claim 2, wherein said oligonucleotide is an oligonucleotide primer for a DNA elongation reaction.
The invention of claim 5, which has been accomplished to achieve the aforementioned object, relates to the oligonucleotide according to claim 2, wherein said oligonucleotide is an oligonucleotide probe a portion of which is modified or labeled with a detectable marker.
The invention of claim 6, which has been accomplished to achieve the aforementioned object, relates to the oligonucleotide according to claim 2, wherein said oligonucleotide is a synthetic oligonucleotide in which a portion of its base(s) is (are) modified without impairing the function of said oligonucleotide as an oligonucleotide probe.
The oligonucleotides of the present invention, which have been accomplished to achieve the aforementioned object, are oligonucleotides that complementarily bind in a specific manner to intramolecular structure-free regions of the target RNA in the aforementioned RNA amplification, and they are capable of binding specifically to the target RNA without the heat denaturation described above. In this manner, the present invention provides oligonucleotides that bind to intramolecular structure-free regions of the GII type SRSV RNA at a relatively low and constant temperature (35-50xc2x0 C., and preferably 41xc2x0 C.), which are useful for specific cleavage, amplification, detection or the like of GII type SRSV RNA. More specifically, the present invention relates to an oligonucleotide primer which cleaves the target RNA mentioned above at specific site, an oligonucleotide primer for amplifying the above target DNA with PCR, an oligonucleotide primer for amplifying the above target DNA with NASBA or the like, and an oligonucleotide probe for detecting the target nucleic acid without or after these amplifications, thereby accomplishes rapid and highly sensitive detection.
SEQ ID Nos. 2 through 9 illustrate examples of the oligonucleotides of the present invention useful in cleavage, amplification, detection or the like of RNA derived from GII type SRSV. In this connection, RNA derived from GII type SRSV also includes RNA that has been produced by using these genes as templates. Although each of the oligonucleotide of the present invention may include entire base sequence of any of SEQ ID Nos.2 to 9, since 10 contiguous bases are adequate for specific binding to GII type SRSV, these oligonucleotides can be oligonucleotides comprising at least 10 contiguous bases of the described sequences.
The oligonucleotides of the present invention can be, for example, used as an RNA-cleavable probe. Cleavage of a target RNA at a specific site can be accomplished by hybridizing the oligonucleotide of the present invention to a single-stranded target RNA, and then exposing it to an enzyme which cleaves only the RNA moieties of the heteronucleic double-stranded RNA-DNA. As for this enzyme, those which are known to have common ribonuclease H activity can be used.
The oligonucleotides of the present invention can be used, for example, as oligonucleotide primers for nucleic acid amplification. If a nucleic acid amplification method is carried out using the oligonucleotide of the present invention as the primer, only the target nucleic acid, namely nucleic acids of the GII type SRSV, can be amplified. Although examples of amplification methods include PCR, LCR, NASBA and 3SR, nucleic acid amplification methods that can be carried out at a constant temperature such as LCR, NASBA and 3SR are particularly preferable. GII type SRSV can be detected by detecting the amplification product by various methods. In this case, any of the above oligonucleotides other than the oligonucleotide used in the amplification may be used as probes, and the fragment of the amplified specific sequence can be confirmed by electrophoresis or the like.
The oligonucleotides of the present invention can be used as probes by, for example, modifying its portion or labeling it with a detectable marker. When detecting the target nucleic acid, the oligonucleotide of the present invention labeled with the detectable marker may be hybridized to a single-stranded target nucleic acid, after which the hybridized probe can be detected via the marker. The marker detection may be carried out by a method suitable for the particular marker and, for example, when using an intercalator fluorescent dye for labeling the oligonucleotide, a dye with the property of exhibiting increased fluorescent intensity by intercalation in the double-stranded nucleic acid comprising the target nucleic acid, and the oligonucleotide probe, may be used in order to allow easy detection of only the hybridized probe without removal of the probe that has not hybridized to the target nucleic acid. When using a common fluorescent dye as the marker, the marker may be detected after removal of the probe that has not hybridized to the target nucleic acid. For the detection, the target nucleic acid in the sample is preferably amplified to a detectable amount by a nucleic acid amplification method such as PCR, NASBA or 3SR method, among which isothermal nucleic acid amplification methods such as the NASBA and 3SR methods are most preferable. When incorporating the nucleotide-labeled probe in the reaction solution during the amplification, it is especially preferable to modify the probe by, for example, adding glycolic acid to the 3xe2x80x2-end so that the probe will not function as a nucleotide primer.
The invention of claim 7, which has been accomplished to achieve the aforementioned object, relates to a GII type SRSV RNA amplification process in which the specific sequence of said GII type SRSV RNA present in a sample is used as a template for synthesis of a cDNA employing an RNA-dependent DNA polymerase, the RNA of the formed RNA/DNA hybrid is decomposed by Ribonuclease H to produce a single-stranded DNA, said single-stranded DNA is then used as a template for production of a double-stranded DNA having a promoter sequence capable of transcribing RNA comprising said specific sequence or the sequence complementary to said specific sequence employing a DNA-dependent DNA polymerase, said double-stranded DNA produces an RNA transcription product in the presence of an RNA polymerase, and said RNA transcription product is then used as a template for cDNA synthesis employing said RNA-dependent DNA polymerase, wherein said RNA amplification process being characterized by employing a first primer comprising at least 10 contiguous bases, of any of the sequences listed as SEQ. ID. No.20 to No.24, which has a sequence homologous to a portion of said GII type SRSV RNA to be amplified, and a second primer comprising at least 10 contiguous bases, of any of the sequences listed as SEQ. ID. No.25 to No.31, which has a sequence complementary to a portion of said GII type SRSV RNA sequence to be amplified (where either or both the first and second primers include the RNA polymerase promoter sequence at their 5xe2x80x2 end).
The invention of claim 8, which has been accomplished to achieve the aforementioned object, relates to the process of claim 7, wherein said RNA amplification process is carried out in the presence of an oligonucleotide probe capable of specifically binding to the RNA transcription product resulting from the amplification and labeled with an intercalator fluorescent pigment, and changes in the fluorescent properties of the reaction solution are measured (with the proviso that said labeled oligonucleotide is different from said first oligonucleotide and said second oligonucleotide).
The invention of claim 9, which has been accomplished to achieve the aforementioned object, relates to the detection method of claim 8, characterized in that said probe is designed so as to complementarily bind with at least a portion of the sequence of the RNA transcription product, and the fluorescent property changes relative to that of a situation where a complex formation is absent.
The invention of claim 10, which has been accomplished to achieve the aforementioned object, relates to the detection method of claim 9, characterized in that said probe comprises at least 10 contiguous bases of any of the sequences listed as SEQ. ID. No. 32 to No. 35 or its complementary sequence.
The present invention provides a nucleic acid amplification process for amplification of GII type SRSV RNA in a sample, and a detection method for RNA transcription products obtained by the nucleic acid amplification process. The amplification process of the invention includes the PCR, NASBA and 3SR methods, but is preferably a constant temperature nucleic acid amplification method such as the NASBA or the 3SR methods whereby GII type SRSV-specific RNA sequences are amplified by the concerted action of reverse transcriptase and RNA polymerase (a reaction under conditions in which reverse transcriptase and RNA polymerase act in concert).
For example, the NASBA method is an RNA amplification process in which the specific sequence of GII type SRSV RNA present in a sample is used as a template for synthesis of a cDNA employing an RNA-dependent DNA polymerase, the RNA of the formed RNA/DNA hybrid is decomposed by Ribonuclease H to produce a single-stranded DNA, the single-stranded DNA is then used as a template for production of a double-stranded DNA having a promoter sequence capable of transcribing RNA comprising the specific sequence or the sequence complementary to the specific sequence employing a DNA-dependent DNA polymerase, the double-stranded DNA produces an RNA transcription product in the presence of an RNA polymerase, and the RNA transcription product is then used as a template for cDNA synthesis employing the RNA-dependent DNA polymerase, and the process of the present invention is characterized by employing a first primer comprising at least 10 contiguous bases of any of the sequences listed as SEQ. ID. No. 20 to No. 24 which has a sequence homologous to a portion of the GII type SRSV RNA, and a second primer comprising at least 10 contiguous bases of any of the sequences listed as SEQ. ID. No. 25 to No. 31, which has a sequence complementary to a portion of the GII type SRSV RNA sequence to be amplified (where either or both the first and second primers include the RNA polymerase promoter sequence at their 5xe2x80x2 region).
While there are no particular restrictions on the RNA-dependent DNA polymerase, the DNA-dependent DNA polymerase and the Ribonuclease H, AMV reverse transcriptase which has all of these types of activity is preferred. The RNA polymerase is also not particularly restricted, but T7 phase RNA polymerase and SP6 phage RNA polymerase are preferred.
In this amplification process, there is added an oligonucleotide which is complementary to the region adjacent and overlapping with the 5xe2x80x2 end of the specific sequence region (bases 1 to 10) of the GII type SRSV RNA sequence, and the GII type SRSV RNA is cleaved (with Ribonuclease H) at the 5xe2x80x2 end region of the specific sequence to prepare the initial template for nucleic acid amplification, thereby allowing amplification of GII type SRSV RNA without the specific sequence at the 5xe2x80x2 end. The oligonucleotide used for this cleaving may, for example, be any of those of SEQ. ID. No. 25 to No. 31 (provided that it differs from the ones used as the first oligonucleotide in the amplification process). The cleaving oligonucleotide is preferably chemically modified (for example, aminated) at the 3xe2x80x2 hydroxyl in order to prevent an extension reaction at the 3xe2x80x2 end.
The RNA amplification product obtained by the aforementioned nucleic acid amplification process may be detected by a known detection method but, preferably, the amplification process is carried out in the presence of an oligonucleotide probe labeled with an intercalator fluorescent pigment, while measuring the changes in the fluorescent properties of the reaction solution. The oligonucleotide probe will typically be the one wherein the intercalator fluorescent pigment is bonded to a phosphorus atom in the oligonucleotide by way of a linker. With this type of suitable probe, formation of a double strand with the target nucleic acid (complementary nucleic acid) causes the intercalator portion to intercalate in the double-stranded portion resulting in a change in the fluorescent property, so that no separatory analysis is necessary (Ishiguro, T. et al. (1996), Nucleic Acids Res. 24(24) 4992-4997).
The probe sequence is not particularly restricted so long as it has a sequence complementary to at least a portion of the RNA transcription product, but it is preferably a sequence comprising at least 10 contiguous bases of the sequences listed as SEQ. ID. Nos.32 to No.35. Also, chemical modification (for example, glycolic acid addition) at the 3xe2x80x2 end hydroxyl group of the probe is preferred in order to prevent an extension reaction with the probe as a primer.
Accordingly, it is possible to amplify and detect RNA comprising the same sequence as the specific sequence of GII type SRSV RNA in a single tube at a constant temperature and in a single step, thus facilitating its application for automation.